Publication Date

2017

Document Type

Dissertation

Committee Members

Steven Berberich (Committee Member), David Cool (Committee Member), Michael Leffak (Advisor), Mill Miller (Committee Member), Yong-jie Xu (Committee Member)

Abstract

Imperative to genomic stability is the ability of the cell to repair damaged DNA which can occur from numerous endogenous byproducts of metabolism or exogenous components from the environment. The Fanconi anemia pathway is a DNA repair mechanism used by human cells to resolve multiple forms of DNA damage including interstrand crosslinks (ICL). Fanconi anemia (FA) is an autosomal recessive inherited disorder characterized by genome instability, developmental abnormalities, cancer predisposition and bone marrow failure. FA is attributed to a mutations in at least 18 genes (FANCA-FANCT) that play a concerted role in DNA repair. FANCT is the latest discovery in the FA pathway and is a UBE2T ubiquitin conjugase. An FA patient with biallelic germline mutations in FANCT presented with the classical symptoms of FA with the exception of hematopoietic indications. Both mutations, a maternal duplication and a paternal deletion of exons 2-6, were AluY mediated. Upon further evaluation it was determined that the FA patient had a reversion of the maternal duplication back to a WT allele which restored FANCT function in the hematopoietic lineage. The genomic reversion was also attributed to an Alu mediated recombination (AMR) event. The factors that led to the AMR event were unknown and provided reason to investigate further. The promise of harnessing the mechanism to utilize a genomic duplication reversion could be of importance for therapeutic interventions. No current human cell line models were sufficient to address what factors are involved in the FANCT reversion, so a novel Dual Fluorescence (DF) model system was created in a HeLa cell line to test the hypothesis that Alu mediated homology directed repair is sufficient to explain genetic reversion of a partially duplicated FANCT locus. The DF model system emulates elements of the FANCT locus using partial intronic sequences and Alu elements found in the patient’s duplicated FANCT locus. Key to the model system design is the incorporation of TOM and eGFP fluorescent marker genes and an I-Sce1 recognition site into the ectopic locus. I-Sce1 is a rare endonuclease found in yeast, with no known recognition site found in humans and when expressed in the DF cells will cause a targeted DNA double strand break. The fluorescent marker genes are separated and flanked by Alu elements. After an I-Sce1 induced DSB, it is possible that AMR will occur and eliminate one of the fluorescent marker genes, thereby shifting fluorescence of the cell. This shift in fluorescence can be detected by microscopy or flow cytometry. To test whether Alu mediated homology directed repair is sufficient to explain genetic reversion of a partially duplicated FANCT locus, multiple DF cell lines were created that vary in; location of the I-Sce1 recognition site, number of Alu elements and the presence of repetitive DNA sequences. Flow cytometry of the DF cells after an I-Sce1 mediated DSB, reveals that recombination of the proximal Alu elements is the most likely repair outcome. Further evaluation of DF cell line recombinants by Flow Assisted Cell Sorting (FACS) and DNA sequencing disclose that the model system can differentiate between homology and non-homology directed repair. To test if Alu mediated homology directed repair can facilitate a genetic reversion, canonical DNA repair proteins in homology directed repair (HDR) and non-HDR pathways were impaired. The results of this experiment provide evidence that only HDR pathways support the genetic reversion. Upon evaluation of a FANCT null cell line in the DF model and the well-established U2-OS GFP model, both models indicate FANCT is involved in homology directed repair. The unique DF model system was able to determine that FANCT is also involved in non-HDR. These experiments provides evidence for the novel finding of FANT’s role in HDR and non-HDR outside of ICL r...

Page Count

132

Department or Program

Biomedical Sciences

Year Degree Awarded

2017


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